Short Forms of Ste20-related Proline/Alanine-rich Kinase (SPAK) in the Kidney Are Created by Aspartyl Aminopeptidase (Dnpep)-mediated Proteolytic Cleavage

Markadieu, Nicolas; Rios, Kerri; Spiller, Benjamin W.; McDonald, W. Hayes; Welling, Paul Pa; Delpire, Eric

doi:10.1074/jbc.m114.604009

Cited by 18 publications

(24 citation statements)

References 47 publications

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“…We recently reported that the renal aspartyl aminopeptidase DNPEP exerts proteolytic cleavage activity against SPAK but not OSR1 (Markadieu et al. ). The sites of cleavage were located within the proline/alanine‐rich region but also within the kinase domain.…”

Section: Discussionmentioning

confidence: 99%

DNPEP is not the only peptidase that produces SPAK fragments in kidney

Koumangoye

Delpire

2017

Physiological Reports

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SPAK (STE20/SPS1‐related proline/alanine‐rich kinase) regulates Na+ and Cl− reabsorption in the distal convoluted tubule, and possibly in the thick ascending limb of Henle. This kinase phosphorylates and activates the apical Na‐Cl cotransporter in the DCT. Western blot analysis reveals that SPAK in kidney exists as a full‐length protein as well as shorter fragments that might affect NKCC2 function in the TAL. Recently, we showed that kidney lysates exerts proteolytic activity towards SPAK, resulting in the formation of multiple SPAK fragments with possible inhibitory effects on the kinase. The proteolytic activity is mediated by a Zn2+ metalloprotease inhibited by 1,10‐phenanthroline, DTT, and EDTA. Size exclusion chromatography demonstrated that the protease was a high‐molecular‐weight protein. Protein identification by mass‐spectrometry analysis after ion exchange and size exclusion chromatography identified multiple proteases as possible candidates and aspartyl aminopeptidase, DNPEP, shared all the properties of the kidney lysate activity. Furthermore, recombinant GST‐DNPEP produced similar proteolytic pattern. No mouse knockout model was, however, available to be used as negative control. In this study, we used a DNPEP‐mutant mouse generated by EUCOMM as well as a novel CRISPR/cas9 mouse knockout to assess the activity of their kidney lysates towards SPAK. Two mouse models had to be used because different anti‐DNPEP antibodies provided conflicting data on whether the EUCOMM mouse resulted in a true knockout. We show that in the absence of DNPEP, the kidney lysates retain their ability to cleave SPAK, indicating that DNPEP might have been misidentified as the protease behind the kidney lysate activity, or that the aspartyl aminopeptidase might not be the only protease cleaving SPAK in kidney.

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Section: Discussionmentioning

confidence: 99%

DNPEP is not the only peptidase that produces SPAK fragments in kidney

Koumangoye

Delpire

2017

Physiological Reports

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“…Consistent with previous reports of SPAK protein expression in mice (McCormick et al, ; Simon, Nelson‐Williams, et al, ), we observed two prominent clusters of SPAK isoforms in mouse kidneys, including a cluster of large molecular weight isoforms running between 55 and 60 kDa, and smaller molecular weight isoforms closer to 45 kDa. The assignment of specific protein bands to specific mouse SPAK isoforms varies from study to study, but the general consensus remains that the larger molecular weight isoforms correspond to catalytically active SPAK, while the smaller molecular weight isoforms correspond to catalytically inactive SPAK in mice (Figure d) (Markadieu et al, ; McCormick et al, ; Simon, Nelson‐Williams, et al, ; Wade et al, ).…”

Section: Resultsmentioning

confidence: 99%

Identification and characterization of alternative STK39 transcripts within human and mouse kidneys reveals species‐specific regulation of blood pressure

et al. 2020

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STK39 encodes a serine threonine kinase, SPAK, which is part of a multi‐kinase network that determines renal Na+ reabsorption and blood pressure (BP) through regulation of sodium‐chloride co‐transporters in the kidney. Variants within STK39 are associated with susceptibility to essential hypertension, and constitutively active SPAK mice are hypertensive and hyperkalemic, similar to familial hyperkalemic hyperkalemia in humans. SPAK null mice are hypotensive and mimic Gitelman syndrome, a rare monogenic salt wasting human disorder. Mice exhibit nephron segment‐specific expression of full length SPAK and N‐terminally truncated SPAK isoforms (SPAK2 and KS‐SPAK) with impaired kinase function. SPAK2 and KS‐SPAK function to inhibit phosphorylation of cation co‐transporters by full length SPAK. However, the existence of orthologous SPAK2 or KS‐SPAK within the human kidney, and the role of such SPAK isoforms in nephron segment‐specific regulation of Na+ reabsorption, still have not been determined. In this study, we examined both human and mouse kidney transcriptomes to uncover novel transcriptional regulation of STK39. We established that humans also express STK39 transcript isoforms similar to those found in mice but differ in abundance and are transcribed from human‐specific promoters. In summary, STK39 undergoes species‐specific transcriptional regulation, resulting in differentially expressed alternative transcripts that have implications for the design and testing of novel SPAK‐targeting antihypertensive medications.

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“…Two of these isoforms that have been discovered in the kidney differ from full length SPAK (~60kDa) in predicted molecular weight and kinase activity; the first isoform SPAK2 (~49kDa) is missing part of the N-lobe of the kinase domain and presumed to be kinase impaired, while the second isoform KS-SPAK (~34kDa) is solely kidney specific and kinase inactive as the entire kinase domain is missing [31]. Note that, as an alternative mechanism to the downstream promoter, the role of a protease has also been proposed as a mechanism for producing the short KS-SPAK isoform [77]. As the CCT domain is intact in these isoforms it is presumed that they compete with full length SPAK and OSR1 for RFxV docking sites, thus inhibiting CCC activity.…”

Section: Role Of Spak In Human Physiology and Diseasementioning

confidence: 99%

Pharmacological targeting of SPAK kinase in disorders of impaired epithelial transport

Zhang

Karimy

Delpire

et al. 2017

Expert Opinion on Therapeutic Targets

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Introduction: The mammalian SPS1-related proline/alanine-rich serine-threonine kinase SPAK (STK39) modulates the transport across and between epithelial cells in response to environmental stimuli such osmotic stress and inflammation. Research over the last decade has established a central role for SPAK in the regulation of ion and water transport in the distal nephron, colonic crypts, and pancreatic ducts, and has implicated deregulated SPAK signaling in NaCl-sensitive hypertension, ulcerative colitis and Crohn’s disease, and cystic fibrosis. Areas covered: We review recent advances in our understanding of the role of SPAK kinase in the regulation of epithelial transport. We highlight how SPAK signaling - including its upstream Cl--sensitive activators, the WNK kinases, and its downstream ion transport targets, the cation- Cl--cotransporters contribute to human disease. We discuss prospects for the pharmacotherapeutic targeting of SPAK kinase in specific human disorders that feature impaired epithelial homeostasis. Expert opinion: The development of novel drugs that antagonize the SPAK-WNK interaction, inhibit SPAK kinase activity, or disrupt SPAK kinase activation by interfering with its binding to Μ025α/β could be useful adjuncts in essential hypertension, inflammatory colitis, and cystic fibrosis.

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Short Forms of Ste20-related Proline/Alanine-rich Kinase (SPAK) in the Kidney Are Created by Aspartyl Aminopeptidase (Dnpep)-mediated Proteolytic Cleavage

Cited by 18 publications

References 47 publications

DNPEP is not the only peptidase that produces SPAK fragments in kidney

DNPEP is not the only peptidase that produces SPAK fragments in kidney

Identification and characterization of alternative STK39 transcripts within human and mouse kidneys reveals species‐specific regulation of blood pressure

Pharmacological targeting of SPAK kinase in disorders of impaired epithelial transport

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